Abstract Formation of neural circuits requires dynamic tissue reorganization comprised of axon elongation to contact targets, presynaptic terminal growth, and dendritic and axonal pruning, which are mediated by multiple cell types. Defining the precise molecular mechanisms of neural circuit formation during early development provides insight into the pathologies of diseases where neural circuit formation goes awry. We utilize the calyx of Held (CH) nerve terminal to study the molecular mechanisms of neural circuit formation because of its rapid growth period (48-72 hours), large size and the clear establishment of a mono-innervation endpoint onto the postsynaptic population of principal neurons in the medial nucleus of the trapezoid body (MNTB). I propose to undertake an extensive RNA-sequencing analysis at the single cell level in the MNTB during the maturation of this neural circuit. This will elucidate novel signaling pathways that regulate the maturation of this neural circuit and assign components of these pathways to a specific cell type to allow for subsequent in vivo perturbations of these pathways. My hypothesis is that key signaling molecules which regulate CH growth and morphological refinement are of both neuronal and glial cell origin and intercellular signaling is then transduced through differential neuronal and glial receptors to regulate the maturation of this system. These findings will allow for follow-up studies of neuron-glial, neuron-neuron and glial-glial communication and functional studies of selective perturbations to this trans-cellular communication. Secondly, I propose to characterize the temporal and spatial dynamics of key elements of signaling pathways with single molecule fluorescent in situ hybridization (smFISH). This analysis will provide quantitative information on mRNA levels in specific cell types/subtypes at different developmental ages and will also offer spatial information of where select neuronal mRNAs are located along the tonotopic axis and which mRNAs reside locally within CH terminals. This information will provide impactful opportunities to understand disorders where neural circuit formation goes awry, such as Autism Spectrum Disorder (ASD) and schizophrenia. Studies focusing on neural circuit formation in the MNTB are of significant clinical relevance because ASD is often accompanied by perturbations in the cellular organization of the auditory brainstem nuclei, giving rise to an auditory phenotype and resulting in communication deficits.